CROHN'S DISEASE EPITHELIAL CELLS EXHIBIT SUSTAINED INFLAMMATORY PROGENITOR CELL STATES

Introduction: Intestinal epithelial stem cells (ISCs) are essential for maintenance of the epithelial barrier, yet their role in Crohn’s disease (CD) is not understood. One impediment to epithelial-targeted therapies for Crohn’s disease is a paucity of data demonstrating whether fundamental differences exist between ISCs at homeostasis and in disease. Prior work from us and others showed a decrease in organoid formation efficiency in patients with inflammatory bowel disease, including Crohn’s disease, suggesting putative disease related ISC deficiencies. We hypothesize that aberrant ISC gene expression and function contributes to sustained inadequate epithelial barrier healing in Crohn’s disease.

Methods: We performed single cell RNA sequencing (scRNA-seq) and single-nucleus ATAC sequencing on ascending colon biopsies from pediatric patients with Crohn’s disease (CD, n=11) and age-matched healthy control subjects (n=12) using the 10x Chromium Next GEM Single Cell 3 v3.1. We performed Uniform Manifold Approximation and Projection (UMAP), Monocle clustering, trajectory and receptor-ligand interaction analyses on scRNA-seq data and evaluated chromatin accessibility in snATAC-seq data. Colonoids were generated from the same set of biopsies followed by organoid formation efficiency (OFE) assays, histology, flow cytometry, and co-culture with primary human macrophages.

Results: ISCs were reduced in biopsies from CD versus control subjects (7.9±5.5% vs 13.7±8.5%, P=0.077; Fig. 1A), and the OFE was significantly reduced in colonoids from CD (5±2.7% vs 12±5.4%, P= 0.028; Fig. 1B,C). Conversely, we observed the presence of a novel, Crohn’s disease-specific inflammatory secretory progenitor (ISP) cluster of cells (11.6±16.9% vs 0.4±0.7%, P=0.03; Fig.2) marked by expression of OLFM4, HLA-DRA, LCN2, REG1A, CD74, PLA2G2A transcripts that re-emerged in colonoid culture when stimulated with a Crohn’s disease-relevant cytokine cocktail or co-culture with primary human macrophages. Trajectory analyses revealed that ISPs are related to goblet cells that retain expression of a subset of ISP marker genes. Mechanistically, snATAC-seq data showed that ISCs from Crohn’s biopsies exhibit enhanced chromatin accessibility, particularly around ISP gene loci, compared to control biopsies.

Conclusions: We defined a high-resolution epithelial ISC atlas in children with Crohn’s disease, including a novel, disease-associated secretory progenitor cell state that persists into the goblet lineage. Epigenomic and functional data in colonoids support the premise that in Crohn’s disease ISCs display persistent inflammatory states that may contribute to impaired mucosal healing in patients. Defining the mechanisms by which ISPs emerge will provide a new level of understanding of epithelial pathogenesis in Crohn’s disease and potential new therapeutic strategies.